Our previous studies have demonstrated the rapidity with which platelets and leukocytes accumulate in ischemic myocardium, bringing with them enzymes, precursors and mediators, not normally found in the host tissue. Thus, the ischemic heart becomes endowed with the capacity to generate a variety of mediators which are not found in the normal heart, and can profoundly influence cardiac function. Moreover, interactions between platelets, leukocytes and the blood vessel wall can lead to the formation of unique mediators, e.g. 12,20 diHETE formed by upon the coincubation of platelets and leukocytes, which are not formed by either cell-type alone. The underlying goal is to examine what mediators are formed, in particular, but not exclusively, with respect to arachidonic acid metabolites, by the ischemic heart and to determine their source (invading cells or cardiac tissue) and possible contribution to ischemia-induced changes in myocardial function. The technique of bioassay will represent an important tool in these studies, since we are interested in biological activity (a tautology), and also the ability to recognize or observe the unexpected. This approach has already proved fruitful with the elucidation, isolation and partial purification of a platelet-derived vasolilator factor (possibly a peptide) which is stimulated by two leukocyte-derived mediators, PAF-acether and leukotriene C4. We aim to continue investigations into the nature and role of this factor. Recent studies have highlighted the endothelium as an important source of factors which mediate vasodilation and modulate some vasospastic events. We have recently identified a cytochrome P450-dependent mixed function oxidase, present in the vascular endothelium, which can metabolise arachidonic acid to a vasodilator metabolite(s). In this proposal we intend to isolate and identify this metabolite(s), and determine the contribution of a vascular cytochrome P450-dependent enzyme system to metabolic or drug-induced changes in coronary blood flow and myocardial ischemia. Thus, the major thrust is towards the identification and elucidation of new mediators derived from platelets, leukocytes and blood vessels, which can contribute to vasoregulation, promote or inhibit the aggregation of blood elements, and exacerbate ischemia-induced cardiac dysfunction.